Claudio Denzlinger

Dendritic Cell Immunogenicity Is Regulated by Peroxisome Proliferator-Activated Receptor γ

Dendritic cells (DC) are the most potent APCs known that play a key role for the initiation of immune responses. Ag presentation to T lymphocytes is likely a constitutive function of DC that continues during the steady state. This raises the question of which mechanism(s) determines whether the final outcome of Ag presentation will be induction of immunity or of tolerance. In this regard, the mechanisms controlling DC immunogenicity still remain largely uncharacterized. In this paper we report that the nuclear receptor peroxisome proliferator-activated receptor γ (PPAR-γ), which has anti-inflammatory properties, redirects DC toward a less stimulatory mode. We show that activation of PPAR-γ during DC differentiation profoundly affects the expression of costimulatory molecules and of the DC hallmarker CD1a. PPAR-γ activation in DC resulted in a reduced capacity to activate lymphocyte proliferation and to prime Ag-specific CTL responses. This effect might depend on the decreased expression of costimulatory molecules and on the impaired cytokine secretion, but not on increased IL-10 production, because this was reduced by PPAR-γ activators. Moreover, activation of PPAR-γ in DC inhibited the expression of EBI1 ligand chemokine and CCR7, both playing a pivotal role for DC migration to the lymph nodes. These effects were accompanied by down-regulation of LPS-induced nuclear localized RelB protein, which was shown to be important for DC differentiation and function. Our results suggest a novel regulatory pathway for DC function that could contribute to the regulated balance between immunity induction and self-tolerance maintenance.

Cyclopentenone Prostaglandins Induce Lymphocyte Apoptosis by Activating the Mitochondrial Apoptosis Pathway Independent of External Death Receptor Signaling

15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2) is a naturally occurring cyclopentenone metabolite of PGD2 that possesses both peroxisome proliferator-activated receptor γ (PPAR-γ)-dependent and PPAR-γ-independent anti-inflammatory properties. Recent studies suggest that cyclopentenone PGs may play a role in the down-regulation of inflammation-induced immune responses. In this study, we report that 15d-PGJ2 as well as synthetic PPAR-γ agonists inhibit lymphocyte proliferation. However, only 15d-PGJ2, but not the specific PPAR-γ activators, induce lymphocyte apoptosis. We found that blocking of the death receptor pathway in Fas-associated death domain−/− or caspase-8−/− Jurkat T cells has no effect on apoptosis induction by 15d-PGJ2. Conversely, overexpression of Bcl-2 or Bcl-xL completely inhibits the initiation of apoptosis, indicating that 15d-PGJ2-mediated apoptosis involves activation of the mitochondrial pathway. In line with these results, 15d-PGJ2 induces mitochondria disassemblage as demonstrated by dissipation of mitochondrial transmembrane potential (Δψm) and cytochrome c release. Both of these events are partially inhibited by the broad spectrum caspase inhibitor benzyloxycarbonil-Val-Ala-Asp-fluoromethylketone, suggesting that caspase activation may amplify the mitochondrial alterations initiated by 15d-PGJ2. We also demonstrate that 15d-PGJ2 potently stimulates reactive oxygen species production in Jurkat T cells, and Δψm loss induced by 15d-PGJ2 is prevented by the reactive oxygen species scavenger N-acetyl-l-cysteine. In conclusion, our data indicate that cyclopentenone PGs like 15d-PGJ2 may modulate immune responses even independent of PPAR-γ by activating the mitochondrial apoptosis pathway in lymphocytes in the absence of external death receptor signaling.

Cyclopentenone prostaglandins induce caspase activation and apoptosis in dendritic cells by a PPAR-γ-independent mechanism: Regulation by inflammatory and T cell–derived stimuli

OBJECTIVE Dendritic cells (DC) are professional antigen-presenting cells playing a pivotal role in the induction of immunological responses. There is evidence that DC survival during ongoing immune responses is finite. However, little is known about the mechanisms regulating apoptosis in these cells. Here, we have investigated the effects of the anti-inflammatory cyclopentenone prostaglandins on human monocyte-derived DC. MATERIALS AND METHODS Phenotype of DC was determined by flow cytometry and their allostimulatory potential in mixed leukocyte reaction. Induction of apoptosis in DC was monitored by staining with annexin-V-FITC and propidium iodide, propidium iodide staining of cell nuclei, and fluorimetric assay of caspase activity. Induction of maturation in DC was obtained by stimulation with TNF-alpha, LPS, IFN-gamma, CD40-ligand, or different combinations of these stimuli. PPAR-gamma expression in DC was determined by RT-PCR. RESULTS Exposure of immature DC to cyclopentenone prostaglandins blunted their allostimulatory capacity and skewed their phenotype by downregulating CD1a and costimulatory molecules. These effects were due to activation of caspases and induction of apoptotic cell death in DC by cyclopentenone prostaglandins. Mature DC showed enhanced susceptibility to apoptosis via cyclopentenone prostaglandins as compared with immature DC. Although DC express PPAR-gamma, the corresponding receptor for some of these metabolites, PPAR-gamma activation by a synthetic high-affinity agonist failed to impair DC viability. CONCLUSIONS Cyclopentenone prostaglandins induce apoptosis of human DC by a PPAR-gamma-independent mechanism. Since these compounds are released during an inflammatory event and show anti-inflammatory properties, they may contribute to the downregulation of DC function through apoptotic cell death.

Transendothelial Migration of Hematopoietic Progenitor Cells

Abstract: There is increasing evidence that hematopoietic stem cell mobilization and homing is regulated not only by adhesion molecules and cytokines, but also by chemotactic factors that support transendothelial migration across the bone marrow sinusoidal endothelium. Many receptors for chemotactic mediators belong to the family of G protein‐coupled seven‐transmembrane receptors (7‐TMR). Signaling via G proteins, particularly Gi proteins, results in a chemotactic response of the cells towards a gradient of the corresponding ligand. Recent studies have provided evidence for expression of several 7‐TMR on immature hematopoietic progenitor cells, which potentially mediate chemotactic effects: chemokine receptors (e.g., CXCR4, receptor for stromal cell‐derived factor‐1), receptors for lipid mediators (e.g., the cysteinyl leukotriene receptor cysLT1 and the peripheral cannabinoid receptor cb2), and receptors for neuroendocrine hormones (e.g., the somatostatin receptor sst2). From these studies it can be concluded that migration of hematopoietic progenitor and stem cells is controlled by a variety of chemotactic factors rather than by a single chemokine (e.g., SDF‐1). Trafficking of immature hematopoietic cells may require combined and interactive regulatory functions of these mediators.

The CysLT1 Ligand Leukotriene D4 Supports α4β1- and α5β1-Mediated Adhesion and Proliferation of CD34+ Hematopoietic Progenitor Cells

Cytokines and chemokines control hematopoietic stem and progenitor cell (HPC) proliferation and trafficking. However, the role of nonpeptide mediators in the bone marrow microenvironment has remained elusive. Particularly CysLT1, a G protein-coupled receptor recognizing inflammatory mediators of the cysteinyl leukotriene family, is highly expressed in HPCs. We therefore analyzed the effects of its ligands on human CD34+ HPCs. The most potent CysLT1 ligand, LTD4, rapidly and significantly up-regulated α4β1 and α5β1 integrin-dependent adhesion of both primitive and committed HPC. LTD4-triggered adhesion was inhibited by specific CysLT1 antagonists. The effects of other CysLT1 ligands were weak (LTC4) or absent (LTE4). In serum-free liquid cultures supplemented with various hematopoietic cytokines including IL-3, only LTD4 significantly augmented the expansion of HPCs in a dose-dependent manner comparable to that of peptide growth factors. LTC4 and LTE4 were less effective. In CD34+ cell lines and primary HPCs, LTD4 induced phosphorylation of p44/42 ERK/MAPK and focal adhesion kinase-related tyrosine kinase Pyk2, which is linked to integrin activation. Bone marrow stromal cells produced biologically significant amounts of cysteinyl leukotrienes only when hematopoietic cells were absent, suggesting a regulatory feedback mechanism in the hematopoietic microenvironment. In contrast to antagonists of the homing-related G protein-coupled receptor CXCR4, administration of a CysLT1 antagonist failed to induce human CD34+ HPC mobilization in vivo. Our results suggest that cysteinyl leukotriene may contribute to HPC retention and proliferation only when cysteinyl leukotriene levels are increased either systemically during inflammation or locally during marrow aplasia.

Value of infliximab (Remicade®) in patients with low-risk myelodysplastic syndrome: final results of a randomized phase II trial (EORTC trial 06023) of the EORTC Leukemia Group.

Tumor-necrosis factor alpha activity has been correlated to ineffective erythropoiesis in lower risk myelodysplastic syndromes. Infliximab (Remicade®) is an anti-tumor necrosis factor alpha chimeric antibody that is used in the treatment of patients with rheumatoid arthritis or Crohn’s disease. Forty-six patients with myelodysplastic syndromes and a relatively low risk of developing acute leukemia were included in a randomized phase II study assessing the therapeutic activity of two dosages of infliximab administration (3 mg/kg vs. 5 mg/kg). The primary end point was the response rate. Responses were observed in 3 of 22 patients (13.1%) randomized to the 3 mg/kg arm, versus 0 of 21 patients randomized in the 5 mg/kg arm. According to the statistical design of the current study, neither of the two infliximab dose schedules tested showed sufficient activity as a single agent in this cohort of unselected patients with early myelodysplastic syndrome.

Sulfur-related air pollutants induce the generation of platelet-activating factor, 5-lipoxygenase- and cyclooxygenase-products in canine alveolar macrophages via activation of phospholipases A2

Recent studies have shown that long-term in vivo exposure of dogs to neutral sulfur(IV)/sulfite aerosols induces mild inflammatory reactions, whereas the combination of neutral sulfite with acidic sulfur(VI)/sulfate aerosols evokes less pronounced effects. To understand underlying mechanisms, we studied in vitro the role of lipid mediators in the responses of alveolar macrophages (AMs) to sulfur-related compounds under neutral (pH 7) or moderate acidic (pH 6) conditions. Canine AMs incubated with sulfite at pH 7 released threefold higher amounts of platelet-activating factor than control (P < 0.005). Generation of arachidonic acid, leukotriene B4, 5-hydroxy-eicosatetraenoic acid, prostaglandin E2, thromboxane B2 and 12-hydroxyheptadecatrienoic acid increased twofold (P < 0.0005). However, these metabolites remained unchanged following incubation of AMs with sulfite at pH 6 or with sulfate at pH 7 or pH 6. Mediator release by sulfite-treated AMs at pH 7 stimulated respiratory burst activity of neutrophils. Inhibition of MAPK pathway by PD 98059, of cytosolic (cPLA2) and secretory phospholipases A2 by AACOCF3 and thioetheramide-PC, respectively, reduced sulfite-induced eicosanoid formation in AMs. Sulfite activated cPLA2 activity twofold at pH 7. This mechanism of sulfite-stimulated responses in phospholipid metabolism predicts that chronic exposure to sulfur(IV)/sulfite is associated with a considerable health risk.

Nonpeptide Mediators in the Hematopoietic Microenvironment

Abstract: Migration of hematopoietic stem and progenitor cells (HPCs) is controlled by chemotactic factors released in the hematopoietic microenvironment. In particular, the chemokine SDF‐1, which activates the G protein‐coupled receptor (GPR) CXCR4, plays an important role in progenitor cell mobilization and homing. However, we provide evidence that ligands of other GPRs similarly act on CD34+ hematopoietic progenitors. These ligands comprise non‐peptide mediators, including the cysteinyl‐leukotriene receptor CysLT1, and stimulate migration and integrin‐dependent adhesion of HPCs. Moreover, continuous activation of a GPR by a specific ligand upregulates the responsiveness of other GPRs to their corresponding ligands. These findings suggest that HPC migration may not depend on a single chemokine receptor (e.g., CXCR4). Rather, mobilization and homing of HPCs involve several GPRs, which interact with each other as well as with adhesion molecules. Pharmacological activation and inhibition of the GPR may allow HPC mobilization and homing to be modulated.

Use of veno-venous bypass for resection of malignant pheochromocytoma with vena caval thrombus.

Surgical management of malignant pheochromocytoma with tumor-induced venous obstruction involving the entrance to the right atrium is challenging. The risk of marked hypotension and hemodynamic instability following clamping of the vena cava is increased as a consequence of the sudden decrease in circulating catecholamines. The use of cardiac bypass, however is burdened with additional operating time and coagulopathy. The present report illustrates that veno-venous bypass is a valuable tool during resection of phenochromocytoma with a large vena caval tumor thrombus.

Potential Role of Cysteinyl Leukotrienes in Trafficking and Survival of Hematopoietic Progenitor Cells

In the clinical setting of a stem cell transplantation, progenitor and stem cells within the bone marrow (BM) can be mobilized into the donors’ peripheral blood by administration of hematopoietic growth factors. Since these cells retain the ability to repopulate the hematopoietic compartment of the bone marrow (a process referred to as ‘homing’), they can account for hematopoietic recovery within the patient [1]. Thus both mobilization to the peripheral blood and homing back to the bone marrow crucially depend on the extensive migratory capabilities of hematopoietic stem and progenitor cells (HPC).